Enhancing soil amendment for salt stress using pretreated rice straw and cellulolytic fungi

Rice straw breakdown is sluggish, which makes agricultural waste management difficult, however pretreatment procedures and cellulolytic fungi can address this issue. Through ITS sequencing, Chaetomium globosum C1, Aspergillus sp. F2, and Ascomycota sp. SM2 were identified from diverse sources. Ascomycota sp. SM2 exhibited the highest carboxymethyl cellulase (CMCase) activity (0.86 IU/mL) and filter-paper cellulase (FPase) activity (1.054 FPU/mL), while Aspergillus sp. F2 showed the highest CMCase activity (0.185 IU/mL) after various pretreatments of rice straw. These fungi thrived across a wide pH range, with Ascomycota sp. SM2 from pH 4 to 9, Aspergillus sp. F2, and Chaetomium globosum C1 thriving in alkaline conditions (pH 9). FTIR spectroscopy revealed significant structural changes in rice straw after enzymatic hydrolysis and solid-state fermentation, indicating lignin, cellulose, and hemicellulose degradation. Soil amendments with pretreated rice straw, cow manure, biochar, and these fungi increased root growth and soil nutrient availability, even under severe salt stress (up to 9.3 dS/m). The study emphasizes the need for a better understanding of Ascomycota sp. degradation capabilities and proposes that using cellulolytic fungus and pretreatment rice straw into soil amendments could mitigate salt-related difficulties and improve nutrient availability in salty soils.


Qualitative screening of cellulase-producing isolate and plant growth promoting activities
All 16 fungal isolates underwent screening for endoglucanase activity on CMC agar plates using the Congo red technique.Approximately 8 isolates exhibited zones of hydrolysis on the CMC agar plates, indicating a positive result for cellulase activity (Table 2).Additionally, these isolates were assessed for various plant growthpromoting activities, including phosphate solubilization, potassium solubilization, and indole-3-acetic acid (IAA) production.Isolate SM2, SM4, and SH1 demonstrated the capability to dissolve phosphate, while only isolate SM2 exhibited the ability to solubilize potassium.Regarding IAA production, all isolates exhibited a range of IAA production levels, spanning from 0.03 to 8.36 µg/mL.This indicates that the majority of isolates possessed the capacity to produce IAA, a valuable plant growth-promoting hormone.

The effect of varying pH on isolate growth
The presented data sheds light on fungal biomass and diameter growth across different pH conditions (pH 4 to 9) for a range of fungal isolates (SM1, SM2, SM4, SM5, C1, and F2).Understanding how pH influences fungal biomass growth is pivotal for assessing fungal adaptability and performance in diverse environments.In Fig. 1, the results revealed the influence of different pH values (4, 5.5, 7, and 9) on the diameter growth of six Filter cake powder (F) 6.48 ± 0.05 5.13 ± 1.61 fungal isolates over 15 days.Isolates SM1, SM2, and SM4 exhibited consistent growth diameters across all tested pH values.These isolates demonstrated no significant differences in their responses to varying pH conditions (P ≤ 0.05).In contrast, isolates C1, F2, and SM5 displayed favorable growth under alkaline conditions (pH 9) when compared to neutral pH (pH 5.5).However, isolated C1 faced inhibition at pH 4. It's evident that fungal biomass increases as the pH transitions from acidic (pH 4) towards neutral (pH 7), with potential variations occurring under alkaline conditions (pH 9) (Fig. 1).Isolates SM1, SM2, and F2 achieved the highest biomass production after 14 days at pH 7 compared to pH 4 and pH 9.Under alkaline conditions (pH 9), isolate C1 and SM5 displayed superior biomass production, while isolate SM4 thrived in acidic conditions.The result suggested   www.nature.com/scientificreports/ that these isolates were in various experimental settings, highlighting their versatility and adaptability to diverse pH environments.

Endoglucanase (CMCase) activity and filter paper (FPase) activity of fungal isolate
In this study, we investigated the enzyme activity of six fungal isolates-C1, F2, SM1, SM2, SM4, and SM5specifically focusing on two key enzymes: CMCase (Carboxymethyl Cellulase) and Fpase (Filter Paper cellulase).These enzymes play pivotal roles in cellulose degradation, an essential process for breaking down complex plant materials.Among them, isolate SM2 displayed the highest CMCase activity, measuring at 0.86, followed by isolates C1 (0.82 IU/mL) and SM1 (0.57 IU/mL) (Fig. 2).In contrast, isolates SM4 (0.24 IU/mL), SM5 (0.25 IU/ mL), and F2 (0.39 IU/mL) exhibited lower CMCase activity.FPase activity also exhibited variability across the fungal isolates.Isolate SM2 demonstrated the highest FPase activity at 1.054 FPU/mL, followed by isolates C1 (1.128 FPU/mL) and SM1 (0.525 FPU/mL).Conversely, isolates SM4 (0.335 FPU/mL), SM5 (0.282 FPU/mL), and F2 (0.636 FPU/mL) displayed lower FPase activity.The distinct capacities of these isolates in cellulose breakdown are highlighted by the variety of CMCase and FPase activities; SM2, C1, and SM1 exhibit notable promise in this regard.Protein production by fungi is of paramount importance as it relates to various cellular and extracellular functions.Fungi utilizes proteins for metabolic processes, enzyme production, and structural components, among other vital functions.Therefore, assessing protein production provides insights into the overall metabolic activity of these isolates.Isolate SM1 exhibited the highest protein production, by 0.151 mg/mL.Close behind was isolate C1, with a protein production level of 0.150 mg/mL.Other isolates, including F2 (0.127 mg/mL), SM2 (0.145 mg/mL, SM5 (0.138 mg/mL), and SM4 (0.068 mg/mL), exhibited varying degrees of protein production (Fig. 2).Fungi with higher protein production capabilities, such as SM1 and C1, are useful for enzyme production, bioremediation, and protein synthesis.

CMCase activity and pH value of pre-treatment rice straw with fungal isolates
The investigation into CMCase activity in submerged rice straw subjected to different pretreatments (5% acetic acid and 2% sodium hydroxide) in conjunction with various fungal isolates has provided intriguing insights into cellulase enzyme activity under diverse conditions (Fig. 3).Overall, both acetic acid and sodium hydroxide www.nature.com/scientificreports/pretreatments demonstrated a notable enhancement in CMCase activity compared to the control condition without any treatment.This enhancement can be attributed to the selective solubilization of hemicellulose and lignin by acetic acid and sodium hydroxide, effectively disrupting their structural components 18,19 .This process results in the removal of these substrates from rice straw, thereby rendering cellulose more accessible to enzymatic degradation.Among the fungal isolates tested, F2, SM1, and SM2 consistently exhibited outstanding enzyme release activity across various conditions.Notably, isolated F2 highlighted the highest CMCase activity, achieving significant levels following NaOH pretreatment (0.185 IU/mL).Isolate SM1 displayed robust cellulase activity, particularly following acetic acid treatment (0.138 IU/mL), surpassing its performance under both NaOHpretreated and normal conditions.This isolate-specific variation underscores the adaptability of these fungi to different pretreatment methods and conditions.It is worth noting that the pH values of each treatment remained stable within a range of 5.67 to 5.95 throughout the study, indicating that variations in CMCase activity were primarily attributed to the pretreatment and fungal isolate combinations rather than significant pH fluctuations.

Cellulose content (%) of rice straw inoculated with fungal isolate
The data in Fig. 4 illustrates the cellulose content (%) in rice straws and fungi substrates subjected to different pretreatment methods (NaOH and acetic acid).Pretreatment with NaOH resulted in a significant reduction in cellulose content across all fungal isolates compared to the control group.The cellulose content ranged from 23.0 to 30.1%.This decrease can be attributed to the ability of NaOH to effectively break down lignin and hemicellulose, which are components that encapsulate cellulose.Acetic acid pretreatment also led to a reduction www.nature.com/scientificreports/ in cellulose content when compared to the control group.The cellulose content ranged from 27.2 to 35.2%.While this pretreatment method is milder than NaOH, it still facilitates the removal of lignin and hemicellulose, making cellulose more accessible for enzymatic degradation.Fungal isolates SM1, SM2, SM4, SM5, C1, and F2 exhibited varying degrees of cellulose content reduction after both pretreatment methods.While isolates SM1, SM2 and C1 exhibited the most significant decrease in cellulose content following both NaOH and acetic acid treatments.These variations underscore the distinct capabilities of each fungal isolate in biomass degradation.The significant reduction in cellulose content after both NaOH and acetic acid pretreatment confirms the effectiveness of these methods in breaking down lignin and hemicellulose.Variability in cellulose content reduction among fungal isolates highlights their unique abilities to interact with pretreated rice straws.

The cellulose degradation of fungi under FTIR spectroscopy
The chemical structural changes of rice straw were determined using FTIR under different pretreatment methods (NaOH and CH 3 COOH) and fungal isolates (SM1, SM2, SM4, SM5, C1 and F2) fermentation.The FTIR spectra of samples were shown in Fig. 5, the functional group change of rice straw was particularly reflected between the absorbance spectra 400 cm −1 and 1800 cm −1 .Although the FTIR spectroscopy of rice straw exhibited the same profile in all treatments (control pretreatment rice straw, pretreatment rice straw fermented with fungal isolate), the intensities of the absorption bands differed.The wide band from 3500-3100 cm −1 was attributed to the O-H stretching of hydrogen bonds in cellulose, hemicellulose, and lignin 20 .Figure 5A revealed the intensity decreased following NaOH and acetic acid pretreatments compared to the control raw rice straw because hemicellulose and lignin from rice straw were partially removed, which might have resulted from the degradation of cellulose.
The 1649 cm −1 peaks presented the H-O-H stretching vibration of adsorbed water in the cellulose structure.The band at 1200 to 1100 cm −1 was often associated with the C-O-H stretching of cellulose and hemicellulose in rice straw, while the reduction in bands showed partial removal of lignin and hemicellulose from rice straw (Fig. 5B,C).In comparison to pretreatment rice straw, pretreatment rice straw inoculated with fungal improves cellulose removal.Furthermore, the band at roughly 1700-1720 cm −1 was often characterized as the C-O of ester linkages, the acetyl group in hemicellulose structure, and the coupling between lignin and hemicellulose 21 .
The reduction in the band following fermentation of NaOH pretreated rice with each fungus isolate shows that these links have degraded and that new chemical groups have formed.Furthermore, the band at 1633 cm −1 was attributed to absorption due to C=O group deformation within the alkyl groups of the lignin side chains, and the higher reduced after rice straw treatment with NaOH and fungi compared to rice straw treated with CH 3 COOH and fungi because the alkali hydrolysis reaction may cause partial lignin structure to change from raw rice straw.
The FTIR spectra results revealed that the alkaline (NaOH 2%) and acid (CH 3 COOH 5%) pretreatment rice straw addition with fungal isolate destroyed a considerable amount of lignin, cellulose, and hemicellulose.All the target fungal isolates contributed to the cellulose breakdown process in rice straw.

Production of soil amendment by selected fungal and organic material
Table 3 showed the concentrations of available nutrients (nitrogen and phosphorus) and organic matter content in the soil after a 30 day incubation period with different soil additions such as rice straw, cow manure, biochar, and fungal.Treatment A6, with CaCO 3 pre-treatment, had the greatest available N concentration on Day 7 (1.46 mg/L) and P concentration on Day 14 (1.75 mg/L).In contrast, treatment T1, the untreated control, had the lowest available N and P concentrations throughout the trial.Treatment A9, which included NaOH pretreatment, had the greatest OM percentage on Day 21 (12.5%) compared to other treatments.Overall, treatments that included pre-treatment with CaCO 3 or NaOH exhibited higher nutritional availability and OM content than untreated and CH 3 COOH-treated samples.

The effect of soil amendment by fungal on rice seedling
According to the experiment's findings on rice seedling growth with soil amendment utilizing solid-state fungal fermentation, pre-treated rice straw, biochar and cow manure, treatments B6 (CaCO 3 pre-treatment) had the greatest germination index at 70% (Table 4).This shows that adding CaCO 3 to the soil amendment improved seed germination and early seedling development.Furthermore, B9 (NaOH pre-treatment) had the largest root length (6.66 cm) and shoot length (18.6 cm), outperformed the control treatment by 140% and 22%, respectively, demonstrating improved root system growth even under intense salt stress conditions (Fig. 6).In terms of pH and EC values, B9 significantly increased soil pH, revealing alkalization of the soil but it resulted in the lowest EC.The treated CH 3 COOH rice straw seems like inhibited the rice seedling growth, while the pH was within the range observed for other treatments.Its data gave evidence that the observed effects on plant growth are not directly attributable to changes in soil pH and salinity.Interestingly, the EC values demonstrated unexpected rice seedling development under high salt stress circumstances (EC range from 7.12 to 9.4), despite levels that normally limit yields in many crops.

Discussion
Microbial communities, which include fungi and bacteria, play a significant role in soil ecosystems by regulating nutrient cycling, organic matter breakdown, and soil structure creation, all of which are essential for maintaining soil fertility and sustaining plant growth 19 .The isolation and characterization of microorganisms from varied environments provides prospects for their use in a variety of biotechnological applications.Microorganisms from saline or semi-saline soils, for example, may include salt-tolerant mechanisms or enzymatic activities that might   help remediate saline soils or improve crop salinity tolerance.Similarly, microorganisms isolated from carbon and filter cake powders may break down organic pollutants or facilitate composting processes in agricultural waste management 22 .Notably, fungal isolates such as Chaetomium globosum and Aspergillus sp., derived from carbon and filter cake, have exhibited extraordinary efficacy in rice straw degradation, as proven by our study's excellent results.
Understanding fungal isolates' pH preferences and performance has important implications for environmental management and biotechnology, particularly in terms of maximizing their use in environmental remediation, agriculture, and industrial processes.Our investigation found that the fungal isolates investigated in this study had a wide pH tolerance, spanning from acidic to alkaline.For example, fungal isolates Chaetomium globosum C1 and Aspergillus sp.F2 thrived in alkaline conditions, instead Ascomycota sp.SM2 grew consistently over a pH range of 4 to 9.These results highlight the significance of pH in microbial ecology and bioprocess optimization, highlighting the necessity of taking pH dynamics into account while using their potential, which supported by study of Mustafa et al. 22 .
In our evaluation of rice growth, we propose that our soil amendment, which includes fungal isolates, cow manure, and biochar, might mitigate the harmful effects of salt stress in the environment.Kumari et al. 23 suggested that the use of organic amendments, fungus, biochar, and vermicompost can successfully reduce the negative effects of salinity on plants.Furthermore, Evelin et al. 24 found that mycorrhizal fungus can help plants cope with salt stress.Chaetomium globosum has been found as a salt-tolerant fungus while Aspergillus possesses both salt-tolerant and thermostable qualities, making it beneficial for industrial applications and lignocellulosic consumption 25,26 .Likewise, the use of biochar increases salt buildup at the soil surface, resulting in a more favorable salinity level for plant development below ground while also aiding mechanical desalination 27,28 .Consequently, our study detected an expansion in root development, even under acute salt stress circumstances.
Cellulases are essential for organic matter decomposition and nitrogen cycling in soil conditions 29 .Our investigation highlighted the significant role of cellulase-producing fungal isolates, including fungal isolates Aspergillus F2, Ascomycota sp.SM2, and Chaetomium globosum C1, in efficient cellulose breakdown, as evidenced by CMCase and FPase activity.These isolates show potential for several uses, such as enzyme synthesis and bioremediation, due to their strong protein production capabilities 28,[30][31][32][33] .Chaetomium sp. is commonly found in interior areas and can degrade cellulose-based construction materials, potentially causing structural damage 28 .Despite the limited knowledge offered, this study has demonstrated the potential of Ascomycota sp. in the breakdown of cellulose, a finding corroborated by research published by Pertile et al. 34 .Furthermore, fungal isolates revealed the ability to enhance plant development through nutrient solubilization, and IAA synthesis, highlighting their multifunctionality in soil [35][36][37][38] .
The improvements in CMCase illustrates fungal after acetic acid and sodium hydroxide pretreatment of rice straw illustrate the effectiveness of these methods in improving cellulose accessibility for enzymatic breakdown.These pretreatments preferentially dissolve hemicellulose and lignin of rice straw, increasing cellulose accessibility and allowing for more effective cellulase enzyme activity 33 .NaOH's strong alkaline properties effectively destroy lignin and hemicellulose structures, resulting in a considerable fall in cellulose content.Boodaeng et al. 39 found that pretreatment of rice straw with diluted alkali resulted in higher reducing sugar yields than acid treatment.FTIR spectroscopy revealed considerable structural changes in rice straw throughout pretreatment and SSF by fungi, showing the degradation of lignin, cellulose, and hemicellulose.These findings highlight the potential of fungal isolates such as Ascomycota sp.SM2, Chaetomium globosum C1, and Aspergillus sp.F2 for biomass conversion processes, providing important insights into the efficacy of acid and alkaline pretreatment techniques in biotechnological applications.Despite the technological limitations provided by the low bioavailability of lignocellulosic residues 40 , selecting sufficient pretreatment procedures for these resistant substrates is critical for improving biodegradation efficiency.FTIR research demonstrated that white-rot fungi promote lignin breakdown in cell walls by oxidizing aromatic rings, which supports recent chemical investigations by Bari et al. 41 .
SSF by fungi provides an excellent manufacturing technique for cellulase, delivering reduced costs and greater enzyme titers 42 .In this study, the addition of fungus to soil amendments tended to improve nutrient availability, especially in treatments with NaOH and CaCO 3 -treated rice straw.Fungal-mediated decomposition mechanisms are thought to have accelerated the breakdown of organic materials, resulting in prolonged nutrient release throughout the incubation period.The observed differences in nutrient availability and organic matter content highlight the significance of soil amendment ingredient choices in agricultural activities.Microbial activity is often strong during the early stages of decomposition (Day 7), allowing for the fast breakdown of easily accessible organic molecules.As a result, there is an initial surge in nutrient release as microbial biomass grows and organic matter breakdown accelerates.However, as decomposition advances, the supply of easily decomposable organic matter decreases, reducing microbial activity and corresponding nutrient release rates.This drop is visible in the middle to late stages of the trial (Day 14 onwards).Furthermore, when microbial populations settle and attain a stable state, nutrient mineralization rates may slow, resulting in a reduction in nutrient release into the soil solution.The present investigation reveals that soil amendment using SSF of NaOH or CaCO 3 -pretreated rice straw in combination with fungal inoculation can improve soil fertility and plant development.However, while this strategy enhanced rice development, the resulting pH values may not be ideal for certain plant species that need slightly acidic to neutral soil conditions.The addition of soil additives raised the soil pH since the research began with alkaline soil.Fungi release enzymes and create organic acids including oxalic acid, citric acid, and acetic acid as organic matter decomposes 43 .They also emit alkaline compounds like ammonia and different basic minerals as metabolic byproducts.The total effect of fungal breakdown on soil pH is determined by the balance between the generation of these chemicals and the alkalinity introduced by the NaOH pretreatment procedure.As a result, it is claimed that our soil amendment, which includes NaOH and CaCO 3 -pretreated rice straw and fungal inoculation, may assist in reducing soil acidity by gradually moving the pH to a more neutral Vol:.( 1234567890

Conclusion
In conclusion, the fungal strains Ascomycota sp.SM2, Chaetomium globosum C1, and Aspergillus sp.F2 showed considerable cellulase activity, aiding rice straw decomposition while also stimulating rice plant development.This work sheds light on how fungal isolates, pretreatment techniques, and SSF processes improve soil fertility, promote plant growth, and facilitate biomass breakdown.These discoveries advance our understanding of microbial ecology, biotechnology applications, and the adoption of sustainable farming methods.However, further study is required to determine the impact of various fungal additions on soil remediation, as well as the mechanistic interactions between fungal enzymes, rice straw components and soil dynamics.

Sample collection and microorganisms screening
Saline soil, rice straw was collected from rice field in Daeng Yai, Mueang Khon Kaen district, Khon Kaen province (Thailand).Filter cake powder and biochar were purchased from a market in Khon Kaen province.The geographical coordinates are at 16.4837° N latitude and 102.7480°E longitude.All methods were performed in accordance with relevant guidelines and regulations.Fungal isolation was done by serial dilution method.One gram sample (heavy saline soil, semi-saline soil, filter cake powder, and biochar powder) was suspended in 10 mL sterile distilled water.An aliquot of diluted sample was spread plated onto a potato dextrose (PDA), and lactic acid (85%) was added to the medium (l g/L) to stimulate the growth of fungi, then the agar plates were at 28 ℃ until fungal growth was evident.The differences in morphology microorganisms were collected and purified.The microorganisms were maintained on PDA at 4 ℃ for further study.

Plant growth promoting assay
All isolates were cultured in nutrient broth (NB) medium supplemented with l-tryptophan (500 mg/L) for IAA production test 44 .Salkowski's reagent (2% 0.5 M FeCl 3 in 35% HClO 4 ) was added to the supernatant and read at 536 nm under spectrophotometer.For potassium and phosphates solubilization test, isolates were spotted on Aleksandrov agar medium and National Botanical Research Institute Phosphate (NBRIP) agar medium, respectively 45 , incubated for 5 to 7 days at 28 ℃, then measured the solubility index.

Enzymatic screening
All fungal isolates were screened for visual cellulolytic activity using 2% (w/v) carboxy methyl cellulose (CMC) agar medium.A fungal plug was inoculated aseptically in the center of CMC agar plates and incubated at 28 ℃ for 5-7 days.The plates were examined to produce clear halo zones on CMC plate when it was treated with 1% Congo red dye for 15 min and rinsed with 1 M NaCl.

The effect of pH on fungi growth
Selected fungal isolates were tested for their growth capability across various pH levels.The different pH levels of nutrient agar (NA) medium were prepared and adjusted using hydrochloric acid (0.1 M HCl) or sodium hydroxide (0.1 M NaOH).The pH level was measured using an electrical pH meter before sterilization in an autoclave at 121 ℃.Fungal isolate was measured fungal diameter and biomass after 15 days of incubation.

Endoglucanase assay
The fungal extract was prepared from fungal cultured in a NB medium.After filtering, the fungal biomass was ground by liquid nitrogen.A buffer solution (50 mM sodium citrate (pH 4.8) and 0.02% Triton X-100) was added to grind biomass.The crude enzyme was collected by centrifugation at 8000 rpm at 4 ℃.
Endoglucanase activity was measured according to the procedure described by Ghose et al. 46 .The 3, 5-Dinitro Salicylic Acid (DNS) chemical was used for determining reducing sugars.The reaction of the mixture contained 0.5 mL of 1% CMC in 0.05 M Na-acetate buffer (pH 5) and 0.5 mL of appropriately diluted crude enzyme.The mixture was incubated at 50 ℃ for 1 h.The reducing sugar released was estimated by the addition of 3, 5-dinitrosalicylic acid (DNS) with glucose as standard.The absorbance was read at 540 nm using a spectrophotometer.

Cellulase (FPase) assay
The cellulase activity was determined using the filter paper asset with a Whatman No.1 filter paper strip equivalent to 50 mg of substrate according to Ghosh et al. 46 .The suitable dilution was made up.The reaction mixture contained 1 mL of 0.05 M Na-citrate (pH 5), the filter paper trip, and 0.5 mL of crude enzyme diluted accordingly.The mixture was incubated at 50 ℃ for 1 h, and a 3 mL DNS was added and read at 540 nm by spectrophotometer.The released reducing sugar was estimated based on glucose standards.

CMCase
IU mL = 0.185 enzyme concentration required to release 0.5 mg of glucose . Vol

In vitro cellulase enzyme production under solid-state fermentation (SSF) conditions
Fungal isolates capable of plant growth promotion and cellulose degradation were chosen for enzyme production under SSF conditions.Rice straw was pretreated by two different methods, one with NaOH 2% for 1 h, and another with acetic acid 5% for 1 h.Rice straw was washed until neutral pH and air dried for 2 days.After that, 5 g of rice straw was added into the flask with 50 mL of nutrient medium (2 g (NH 4 ) 2 SO 4 , 2 g KH 2 PO 4 , 0.3 g MgSO 4 , 0.3 g CaCl 2 , 0.5 g NaCl, 0.005 g FeSO 4 , 0.016 g MnSO 4 , 0.017 g ZnCl 2 ).These were autoclaved at 121 ℃ for 15 min.The fungal isolates were grown on NA medium for 5-7 days before harvesting the spores in sterile saline.The obtained spore solution was dispersed in saline, and 1 mL of it was diluted with 9 mL of saline before measuring the optical density (OD) at 600 nm with a spectrophotometer.A hemocytometer was used to determine and modify the spore concentration of the fungal isolates based on their OD value of 1 48 .Subsequently, fungal suspension was added to the flasks and incubated at 28 ℃ for 30 days.For extraction of enzyme activity, 50 mL of citrate buffer (50 mM, pH 4.8) was added and shaken for 30 min.The contents were filtered, and the filtrate was centrifuged at 8000 rpm for 20 min.The final filtrate was collected and measured the enzyme activity following the enzyme assay as mentioned above.

Cellulose content (%)
The cellulose content from rice straw was preformed according to a method described in the study of Luo et al. 49 with some modified.One gram of dry biomass was fluxed for 20 min with 10 mL of 80% acetic acid and 1.5 mL of nitric acid.The mixture was dried in hot air oven at 105 ℃ until it reached a constant weight, and the difference between the initial and final weights was used to calculate the content of cellulose (%)

Cellulose degradation identification through infrared spectroscopy by Fourier transformer (FTIR)
The FTIR spectrum was carried out in a spectrometer FTIR (brand) which has a resolution of 4 cm −1 to characterize the functional groups of the samples, the sample was finely ground and pre-dried KBr before reading by FTIR.The region used for the analysis was 400-4000 cm −1 .

Identification of fungal isolates
Fungal strains were incubated on the NA medium at 28 ℃ for 5-7 days, the colonies were characterized by the morphological method.The total genomic DNA of fungal strains were extracted by the CTAB method 50 .The molecular identification of fungal strains was carried out based on the analysis of internal transcribed spacer (ITS) region sequences using universal primers ITS1.Following sequencing, the Basic Local Alignment Search Tool (BLAST) software was utilized to align the gene sequences of various bacterial strains with bacterial sequences within the National Center for Biotechnology Information (NCBI) databases.

Formula preparation
Rice straw was initially pre-treated by soaking in calcium carbonate (CaCO 3 ), acetic acid (CH 3 COOH), or sodium hydroxide (NaOH) for 20 min to enhance the degradation process.Co-inoculated 3 fungal isolates with 20 ml of spore suspension (fungal spore prepared as follow method of Singh et al. 48) were supplemented to soil amendment.Then, soil amendment was prepared by two formulas as follows the Table 5.The substrates were contained in the cup with a well cover and incubated at 28 ℃ for 30 days.

Characterization of soil amendment substrate by rice straw and fungi
Using a pH meter and an EC meter to measure the pH and EC values of each rice straw amendment formulation were evaluated in water extracts (follow the ratio of 1:1 for pH and 1:5 for EC).The available of nitrogen and phosphorus content were assessed using the Kjeldahl method.The colorimetric method with molybdenum was employed to extract and measure the available phosphate (orthophosphate) in rice straw substrates 51 .

FPase
IU mL = 0.37 enzyme concentration required to release 2 mg of glucose .
Table 5.The formula of soil amendment of rice straw degradation by fungi.

Figure 1 .
Figure 1.Growth (colony expansion) of 6 selected fungal isolates on various pH values on NA medium.Measurements are the mean of three diameter measurements for at least three replication and error bars indicate the standard error of the mean.

Figure 2 .
Figure 2. The enzyme activities and protein concentration produced by fungal isolates.The express as mean value ± SDTEV, the small letters indicated significant differences in values of each fungal isolate (by LSD, P ≤ 0.05).

Figure 3 .Figure 4 .
Figure 3.CMCase activity of fungal isolates from quantitative screening under submerged inoculation in various pre-treatment.The express as mean value ± SDTEV, the small letters indicated significant differences in values of each fungal isolate (by LSD, P ≤ 0.05).

Figure 5 .
Figure 5. FTIR spectra of rice straw in different pretreatment method and various fungal isolate.(A) Control rice straw compared to NaOH or CH 3 COOH pretreatment rice straw; (B) CH 3 COOH pretreatment rice straw with 3 differences fungal isolates; (C) NaOH pretreatment rice straw with 3 differences fungal isolates.

Table 1 .
The pH and EC value of 4 distinct kinds of soil sample.

Table 2 .
Relative enzyme activity index (I CMC ) and plant growth promoted by outstanding isolates.Noted that (-) negative result.

Table 3 .
Soil amendment availability of nitrogen, phosphate, and organic matter with inoculation of co-fungal isolates over 21 and 30 days.To indicate significant differences at the 0.05 level, used (*); to indicate significant differences at the 0.01 level, used (**).Small alphabetic letters indicate significant differences of each treatment by F-test (P ≤ 0.05).

Table 4 .
The effect of soil amendment by pre-treated rice straw and fungal inoculation on rice seedlings.To indicate significant differences at the 0.05 level, used (*); to indicate significant differences at the 0.01 level, used (**).Small alphabetic letters indicate significant differences of each treatment by F-test (P ≤ 0.05).
or slightly alkaline level.This change might have a favorable impact on plant development and overall soil health in agricultural contexts.